A cell's communication with its surroundings is central to numerous physiological functions in both normal and diseased states. It requires that encounters with extracellular ligands can be sensed inside the cell, and conversely, that the strength of extracellular ligand-receptor interactions can be modulated by intracellular processes. Adapter proteins that link the cytoplasmic domains of cell-surface receptors to the cytoskeleton (cytoskeletal membrane anchors) are likely to play a crucial biophysical role in this bidirectional communication, i.e., not only as cell-signaling messengers, but also as structural elements that mediate, and possibly regulate, a cell's response to mechanical stimuli. Accordingly, this application puts forward the paradigm that serial linkages of the form extracellular ligand - transmembrane receptor - cytoskeletal anchor - cortical actin represent functional units that act as key biophysical switchboards in cellular communication. Often such serial linkages are exposed to physical stress, in which case the strength hierarchy of individual molecular interactions, along with the physical properties of the plasma membrane and the actin cortex, provide the mechanistic foundation of their mechanoregulatory behavior. Understanding these complex mechanisms requires an interdisciplinary, nano-to-microscale approach. We propose to systematically dissect the individual contributions of the constituents of two types of serial transmembrane linkages. Aims 1 and 2 will establish the structural behavior of serial linkages involving the integrin LFA-1 and E- and N-cadherins. Aim 1 will focus on extracellular binding by quantifying the dynamics of formation and force-dependent failure of the respective receptor:ligand bonds (using cutting-edge force- probe instruments, i.e., our side-view AFM and optical tweezers). Aim 2 will examine the mechanical behavior and the molecular determinants of the cytoskeletal anchors of these receptors. Combining force probing and fluorescent functional imaging, Aim 3 will investigate how this structural organization correlates with cellula function. For example, we will explore the mechanisms of leukocyte activation by mechano-chemical stimuli. This strategy will open new avenues toward establishing a biologically plausible and physically realistic understanding of these remarkable mechanosignaling paths, and thus also toward novel bottom-up strategies for diagnosis and treatment of diseases. PUBLIC HEALTH RELEVANCE: Although vital processes like the migration of immune cells to sites of inflammation, or the migration, homing, and invasion of cancer cells during metastasis, rely heavily on the regulation of the cells' ability to hold on to each other at one stage and let o at another, it remains poorly understood how a cell, at the opportune moment, instructs the extracellular domains of its docking molecules to let go. Likewise, little is known about how a cell translates external mechanical stimuli into well-defined cytoplasmic responses. This application posits that cytoskeletal membrane anchors are key switchboards in force-sensitive cellular communication. Using innovative biophysical concepts and tools, this project will elucidate mechanoregulatory processes that are inaccessible to existing techniques, and thus provide new insight for future bottom-up strategies to fight cancer, immune defects, and other diseases.